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Melanoma is an aggressive and treatment resistant tumor of the melanocyte lineage. In humans, melanocytes are responsible for skin pigmentation and the tanning response. Pigmentation and the tanning response are controlled by the Melanocortin 1 Receptor (MC1R) which signals through the cAMP cascade. Individuals with defective MC1R often show the Red Hair Color (RHC) phenotype which is marked by red hair, freckling and decreased tanning response. This phenotype and the MC1R RHC genotype confer high risk to skin cancers, including melanoma. Although some of this risk is attributable to increased mutation rates in MC1R RHC cells, we sought to determine whether active MC1R and cAMP signaling could inhibit melanoma growth independently from these effects on mutation. We investigated the activity of MC1R RHC alleles in terms of cAMP signaling and investigated the role of MC1R and cAMP signaling in the growth and proliferation of normal primary melanocytes, melanocytes expressing activated oncogenes and melanoma cell lines. In primary cells, we found that MC1R RHC alleles had defective, but not complete loss of function cAMP signaling. We found that MC1R activity has a mitogenic role in normal melanocytes, but may cooperate with BRaf V600E in causing cell cycle arrest. We show that forskolin treatment slowed the growth of melanoma cell lines in culture. MC1R overexpression, treatment with the MC1R ligand or other small-molecule activators of cAMP signaling caused a delay in progression from G2 into mitosis. This delay was caused by inhibition of the CDK1 phosphatase cdc25B through phosphorylation at PKA sites and was rescued by expression of a cdc25B mutant that could not be phosphorylated by PKA. These data show that MC1R/cAMP signaling can inhibit melanoma growth outside of its role in protecting cells from mutagenesis and suggest that cAMP activators could represent a therapeutic strategy in melanoma.

Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract that has limited treatment options. To gain insight into the pathogenesis of chronic colonic inflammation (colitis), we performed a multiomics analysis that integrated RNA microarray, total protein mass spectrometry (MS), and phosphoprotein MS measurements from a mouse model of the disease. Because we collected all three types of data from individual samples, we tracked information flow from RNA to protein to phosphoprotein and identified signaling molecules that were coordinately or discordantly regulated and pathways that had complex regulation in vivo. For example, the genes encoding acute-phase proteins were expressed in the liver, but the proteins were detected by MS in the colon during inflammation. We also ascertained the types of data that best described particular facets of chronic inflammation. Using gene set enrichment analysis and trans-omics coexpression network analysis, we found that each data set provided a distinct viewpoint on the molecular pathogenesis of colitis. Combining human transcriptomic data with the mouse multiomics data implicated increased p21-activated kinase (Pak) signaling as a driver of colitis. Chemical inhibition of Pak1 and Pak2 with FRAX597 suppressed active colitis in mice. These studies provide translational insights into the mechanisms contributing to colitis and identify Pak as a potential therapeutic target in IBD., Crohn's and Colitis Foundation of America (Research Fellowship), National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant 1122374), Institute for Collaborative Biotechnologies (W911NF-09-0001)

Inflammatory bowel disease (IBD) is a chronic condition driven by loss of homeostasis between the mucosal immune system, the commensal gut microbiota, and the intestinal epithelium. Our goal is to understand how these components of the intestinal ecosystem cooperate to control homeostasis. By combining quantitative measures of epithelial hyperplasia and immune infiltration with multivariate analysis of inter- and intracellular signaling, we identified epithelial mammalian target of rapamycin (mTOR) signaling as a potential driver of inflammation in a mouse model of colitis. A kinetic analysis of mTOR inhibition revealed that the pathway regulates epithelial differentiation, which in turn controls the cytokine milieu of the colon. Consistent with our in vivo analysis, we found that cytokine expression of organoids grown ex vivo, in the absence of bacteria and immune cells, was dependent on differentiation state. Our study suggests that proper differentiation of epithelial cells is an important feature of colonic homeostasis because of its effect on the secretion of inflammatory cytokines., Author summary Chronic inflammation of the gastrointestinal track is the common defect shared by inflammatory bowel diseases (IBDs), such as Crohn’s disease and ulcerative colitis, which affect many people around the world. However, the genetic and physiologic complexities of IBDs have made it difficult to identify therapeutically tractable drivers of disease that can alleviate the symptoms. We reasoned that this complexity is probably originated by a smaller number of dysregulated signaling pathways, and therefore, a “protein-centric” approach would be more suited to identify new therapeutic targets. To this end, in this study we profiled the expression and phosphorylation status of proteins that mediate signaling between and within cells in a mouse model of colitis. We found that hyperactivated mammalian target of rapamycin (mTOR) signaling interferes with the proper differentiation of epithelial cells, which promotes colitis by altering the epithelial inflammatory cytokine secretion in the colon.

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine that can elicit distinct cellular behaviors under different molecular contexts. Mitogen activated protein kinase (MAPK) pathways, especially the extracellular signal-regulated kinase (Erk) pathway, help to integrate influences from the environmental context, and therefore modulate the phenotypic effect of TNF-α exposure. To test how variations in flux through the Erk pathway modulate TNF-α-elicited phenotypes in a complex physiological environment, we exposed mice with different Ras mutations (K-Ras activation, N-Ras activation, and N-Ras ablation) to TNF-α and observed phenotypic and signaling changes in the intestinal epithelium. Hyperactivation of Mek1, an Erk kinase, was observed in the intestine of mice with K-Ras activation and, surprisingly, in N-Ras null mice. Nevertheless, these similar Mek1 outputs did not give rise to the same phenotype, as N-Ras null intestine was hypersensitive to TNF-α-induced intestinal cell death while K-Ras mutant intestine was not. A systems biology approach applied to sample the network state revealed that the signaling contexts presented by these two Ras isoform mutations were different. Consistent with our experimental data, N-Ras ablation induced a signaling network state that was mathematically predicted to be pro-death, while K-Ras activation did not. Further modeling by constrained Fuzzy Logic (cFL) revealed that N-Ras and K-Ras activate the signaling network with different downstream distributions and dynamics, with N-Ras effects being more transient and diverted more towards PI3K-Akt signaling and K-Ras effects being more sustained and broadly activating many pathways. Our study highlights the necessity to consider both environmental and genomic contexts of signaling pathway activation in dictating phenotypic responses, and demonstrates how modeling can provide insight into complex in vivo biological mechanisms, such as the complex interplay between K-Ras and N-Ras in their downstream effects., National Institute of General Medical Sciences (U.S.) (Grant R01-GM088827), National Cancer Institute (U.S.) (U54-CA112967), United States. Army Research Office (Institute for Collaborative Biotechnologies Grant W911NF-09-D-000)